The differentiation of mammalian germ cells involves a complex process of morphogenetic changes. To identify the genes which may be involved in thes processes, we have used a strategy based upon the observation that a region of DNA sequence, called the homeobox, is highly conserved in several genes in Drosophila which have been shown to be important in regulating development and differentiation as well as in the genomes of higher organisms. We isolated the mouse homeobox-containing gene Hox-1 4, determined its molecular structure, and examined its tissue, cellular, and temporal specificity of expression at the level of RNA. We will now characterize the Hox-1 4 protein and determine when it is actually translated during spermatogenic differentiation. Antibodies will be generated against the Hox-1 4 protein product. The antibodies will be used to characterize the Hox-1 4 protein and to determine its intracellular localization. We will also examine the possible role of Hox-1 4 in regulating the expression of other homeobox-containing genes expressed in the testis, such as Hox-1 3, Hox-1 5, Hox-2 6 and Hox-5 1. This will initially involve determining the developmental, temporal, and cellular specificity of expression in the testis of these genes and comparing this pattern of expression to that observed in transgenic mice in which Hox-1 4 expression has been altered. Subsequent studies will be directed to determining if the Hox-1 4 protein binds to specific DNA sequences within Hox-1 4 itself and/or to other homeobox-containing genes expressed in the testis. Finally, we will begin to assess the function of Hox-1 4 in the germ line by generating transgenic mouse lines in which we will manipulate the expression of Hox-1 4 by overexpressing Hox-1 4 in germ cells which normally express the gene and by expressing antisense constructs of Hox-1 4 under the direction of spermatogenic-stage specific promoters. These results will provide new insight into the function of homeobox-containing genes during mammalian germ cell development.